High Twist Expression Research Articles

DIFFERENTIALLY REGULATED MIRNAS BY TWIST1 IN TRIPLE NEGATIVE BREAST CANCER CELLS

Objective: Breast cancer (BC) is the most common cancer in women and the second leading cause of cancer-related deaths. MicroRNAs (miRNAs) are short, non-coding RNA molecules that regulate gene expression post-transcriptionally and play a central role in the dysregulation of gene expression associated with carcinogenesis, cancer cell proliferation and metastasis. Twist1 is a transcription factor that binds to E-box motifs and controls the transcriptional activity of genes as a positive or negative regulator decisive in the cellular mechanisms. Accordingly, Twist1 also regulates the expression of miRNAs that are associated with cancer progression. In present study, we aimed to investigate the expressional changes of possible miRNAs directly regulated by Twist1 in triple negative breast cancer MDA-MB-231 cells. Materials and Methods: In this study, a total of 43 miRNA genes were evaluated that predicted might be associated with triple negative breast cancer. To determine the Twist1-targeted miRNA genes, endogenous high level Twist1 expression was suppressed through the antisense oligonucleotides in MDA-MB-231 TNBC cells. Differential miRNA expression levels were analyzed by real time PCR analysis in Twist1-suppressed cells compare to control. Results: Twist1 suppression leads to an increase in miR-1-1 and miR-210-3p expression, while a decrease in miR-193b-3p, miR-181b-5p, and miR-148a-3p expression. Conclusion: This study shows that the expression levels of certain miRNAs linked to invasion, metastasis, and apoptosis are controlled by Twist1 in triple negative breast cancer cells.

TMOD-03.BRAFV600E MUTATION INDUCES TWIST1 UPREGULATION ASSOCIATED WITH MIGRATION AND CEREBRAL FLUID DISSEMINATION IN GLIOBLASTOMA

Abstract INTRODUCTION BRAFV600E-mutant glioblastoma (GBM) with epithelioid features frequently show cerebrospinal fluid dissemination (CSFD), which results in poor prognoses. BRAF mutation is worth investigating since BRAF or downstream MEK inhibitors show dramatic response for BRAFV600E-mutant GBM including CSFD. This suggests BRAF mutation could play a role for inducing the clinical manifestation of CSFD found in newly diagnosed and recurrent BRAFV600E-mutant GBM. Because, BRAFV600E-mutant GBM often recur with CSFD, elucidating the mechanism by which BRAFV600E-mutant GBM exhibits CSFD is important to improve prognosis. (Objective) To elucidate the molecular mechanism of CSFD involved in BRAFV600E-mutant GBM. METHODS We introduced the heterozygous BRAFV600E mutation in human induced pluripotent stem cells (iPSC) in the context of GBM genetic alterations, CDKN2A/2B, and PTEN deletion, TERT promoter mutation and with EGFRvIII overexpression. We implanted these iPSC-derived neural progenitor cells into immunodeficient mouse brains to establish a BRAFV600E-mutant GBM model. At the same time, we created a model with the same genetic background without BRAFV600E. We compared their gene expression, pathology, and migration patterns to identify BRAFV600E specific characteristics. RESULTS RNA sequencing revealed BRAFV600E-mutant tumors upregulated migration, and downregulated cell-to-cell adhesion genes. TWIST1 was one of the most upregulated genes, and was validated by qPCR. Concomitant with TWIST1 expression we detected downregulation of E-cadherin. The BRAFV600E GBM model histologically showed a cluster-like invasion phenotype, while non-BRAF tumors showed homogenous invasion with typical GBM pathological findings. Immunohistochemical staining showed relatively high TWIST1 expression in cluster-like tumor cells of BRAFV600E tumors. Upon re-engraftment of primary tumor-derived cells, secondary BRAFV600E tumors presented leptomeningeal dissemination. TWIST1 knockdown resulted in a decrease in the migratory ability of our BRAFV600E model in vitro, and is under investigation in vivo. CONCLUSION BRAV600E mutation induces TWIST1 upregulation, and might facilitate migration and CSFD. This work nominates TWIST1 and its regulated genes as targets for BRAFV600E-mutant GBM with CSFD.

The prognostic significance of twist in pancreatic cancer and its role in cancer promotion through the regulation of the immune microenvironment and EMT mechanisms

ObjectivePancreatic cancer has a poor prognosis due to its high malignancy and rapid progression. The limited immunogenicity of pancreatic cancer (PAAD) contributes to its low responsiveness to immunotherapy, yet its underlying mechanism remains poorly understood. As a cancer promoting gene, Twist participates in EMT in various tumors and promotes tumor progression. The interplay between EMT and the tumor microenvironment (TME) emerges as a pivotal factor influencing tumor immunity and response to immunotherapy. Twist therefore has potential as a biomarker for gauging the outcome of tumour immunotherapy.This research aimed to assess the Twist's prognostic significance in PAAD and its relationship to immunotherapy response.MethodsIn this research, transcriptional data and epigenetic alterations of Twist in pancreatic cancer, along with their impact on the prognosis of PAAD patients, were analyzed using databases. Functional enrichment analysis elucidated the biological role of Twist in PAAD. Subsequently, databases including CIBERSORT and TIDE were employed to investigate the association between Twist expression and immune cell infiltration, immune checkpoint genes, and immunotherapy sensitivity within the pancreatic cancer immune microenvironment.Paraffinized specimens from patients with pancreatic ductal adenocarcinoma confirmed by postoperative pathology were selected for Twist expression verification, and the difference was analyzed by Chi-square test; uncontaminated pancreatic cancer cell lines were used for Twist expression verification, and the differences were analyzed by Student t-test.ResultsTwist mRNA expression was notably upregulated in PAAD, positively correlating with gene methylation levels. Analyses of Kaplan–Meier and Cox regression showed a correlation between better overall survival and lower Twist expression. Functional annotation indicated that Twist-associated differentially expressed genes (DEGs) were involved in EMT regulation and acute inflammation. High expression of Twist leads to a significant reduction in the infiltration of anti-tumor immune cells such as Monocytes, NKcellsactivated, and TcellsCD8, further supporting its creation of a typical immunosuppressive microenvironment in pancreatic cancer. Twist expression is positively correlated with the expression of HARVCR2, LAIR1, LGALS3 and other genes, which may be related to the treatment response to immune checkpoint inhibitors (ICIs). TIDE analysis predicts that patients with high expression of Twist will be insensitive to immunotherapy. Twist is significantly over-expressed in pancreatic cancer cell lines and tissues, and is negatively correlated with E-cadhrin expression, but positively correlated with N-cadherin,Snail, and ZEB1.ConclusionHigh Twist expression in PAAD signifies a grim prognosis. Its elevated levels not only contribute to tumor progression through EMT induction but also exert regulatory control over the immune microenvironment, leading to immunosuppression and diminished effectiveness of immunotherapy.

DNA-PKcs/AKT1 inhibits epithelial-mesenchymal transition during radiation-induced pulmonary fibrosis by inducing ubiquitination and degradation of Twist1.

Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive, irreversible lung interstitial disease that develops after radiotherapy. Although several previous studies have focused on the mechanism of epithelial-mesenchymal transition (EMT) in lung epithelial cells, the essential factors involved in this process remain poorly understood. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exhibits strong repair capacity when cells undergo radiation-induced damage; whether DNA-PKcs regulates EMT during RIPF remains unclear. To investigate the role and molecular mechanism of DNA-PKcs in RIPF and provide an important theoretical basis for utilising DNA-PKcs-targeted drugs for preventing RIPF. DNA-PKcs knockout (DPK-/-) mice were generated via the Cas9/sgRNA technique and subjected to whole chest ionizing radiation (IR) at a 20Gy dose. Before whole chest IR, the mice were intragastrically administered the DNA-PKcs-targeted drug VND3207. Lung tissues were collected at 1 and 5 months after IR. The expression of DNA-PKcs is low in pulmonary fibrosis (PF) patients. DNA-PKcs deficiency significantly exacerbated RIPF by promoting EMT in lung epithelial cells. Mechanistically, DNA-PKcs deletion by shRNA or inhibitor NU7441 maintained the protein stability of Twist1. Furthermore, AKT1 mediated the interaction between DNA-PKcs and Twist1. High Twist1 expression and EMT-associated changes caused by DNA-PKcs deletion were blocked by insulin-like growth factor-1 (IGF-1), an AKT1 agonist. The radioprotective drug VND3207 prevented IR-induced EMT and alleviated RIPF in mice by stimulating the kinase activity of DNA-PKcs. Our study clarified the critical role and mechanism of DNA-PKcs in RIPF and showed that it could be a potential target for preventing RIPF.

High Twist expression can be an early event in lip carcinogenesis

AbstractObjectiveActinic cheilitis (AC) is a chronic lesion that usually precedes the onset of squamous cell carcinoma of the lip. Microscopically, AC may exhibit epithelial dysplasia (ED). Twist is a transcription factor that regulates the mesenchymal phenotype through negative E‐cadherin regulation. This study evaluated Twist and E‐cadherin immunoexpression in AC with different ED degrees.Materials and MethodsTwist and E‐cadherin immunoexpression was evaluated semiquantitatively in the epithelium of 86 cases of AC with different ED degrees. Data obtained were submitted to the Mann–Whitney test and Spearman's correlation test.ResultsImmunohistochemical analysis revealed nuclear and cytoplasmic Twist immunoreactivity in all epithelial layers except the cornified layer. In contrast, the membrane and cytoplasmic E‐cadherin immunoreactivity was observed in all epithelial layers except the cornified layer. No significance was observed between the scores of these proteins and the ED degree. It was observed a correlation between total Twist and total (p = 0.030) and membrane (p = 0.014) E‐cadherin and between nuclear Twist and cytoplasmic E‐cadherin (p = 0.030).ConclusionsThe results suggest that the high immunoexpression of Twist is an early lip carcinogenesis event. However, it appears not to influence the progression of ED in AC.

E-CADERIN, N-CADERIN, SLUG, SNAIL, and TWIST contribute to epithelial-mesenchymal transition in salivary gland tumors.

Transcription factors are important in the epithelial-mesenchymal transition process and are possibly related to the development of a more invasive tumor phenotype. Thus, the objective of this study was to analyze the expression and identify the localization of cellular markers related to the epithelial-mesenchymal transition process in salivary gland tumors. The expression and localization of E-CADERIN, N-CADERIN, SLUG, SNAIL, and TWIST were evaluated, using immunohistochemistry, in 48 salivary gland tumors, being 17 pleomorphic adenomas (PA), 14 adenoid cystic carcinomas (ACC), and 17 mucoepidermoid carcinomas (MEC). these proteins were compared to clinical and histopathologic parameters. normal gland tissues were included for immunohistochemical comparisons. ACC and MEC cases showed higher expression of SNAIL compared to PA. MEC showed high expression of SLUG and TWIST. Low expression of N-CADHERIN, SNAIL, and TWIST in ACC was frequent in T3 and T4. High expression of TWIST in MEC was more frequent at age ≥ 40 years A positive correlation was only observed between N-cadherin/SNAIL in ACC, between SNAIL/TWIST in MEC, and between SLUG/TWIST in PA. This study provided insight into EMT-related proteins (E-cadherin, N-cadherin, SNAIL, SLUG, and TWIST) and their contribution to the maintenance of morphogenesis and the development of the salivary gland tumors and showed a positive correlation among N-CADHERIN/SNAIL in ACC and SNAIL/TWIST in MEC.

Research progress of Claudin-low breast cancer.

Claudin-low breast cancer (CLBC) is a subgroup of breast cancer discovered at the molecular level in 2007. Claudin is one of the primary proteins that make up tight junctions, and it plays crucial roles in anti-inflammatory and antitumor responses as well as the maintenance of water and electrolyte balance. Decreased expression of claudin results in the disruption of tight junction structures and the activation of downstream signaling pathways, which can lead to tumor formation. The origin of Claudin-low breast cancer is still in dispute. Claudin-low breast cancer is characterized by low expression of Claudin3, 4, 7, E-cadherin, and HER2 and high expression of Vimentin, Snai 1/2, Twist 1/2, Zeb 1/2, and ALDH1, as well as stem cell characteristics. The clinical onset of claudin-low breast cancer is at menopause age, and its histological grade is higher. This subtype of breast cancer is more likely to spread to lymph nodes than other subtypes. Claudin-low breast cancer is frequently accompanied by increased invasiveness and a poor prognosis. According to a clinical retrospective analysis, claudin-low breast cancer can achieve low pathological complete remission. At present, although several therapeutic targets of claudin-low breast cancer have been identified, the effective treatment remains in basic research stages, and no animal studies or clinical trials have been designed. The origin, molecular biological characteristics, pathological characteristics, treatment, and prognosis of CLBC are extensively discussed in this article. This will contribute to a comprehensive understanding of CLBC and serve as the foundation for the individualization of breast cancer treatment.

The Transcription Factor Twist1 Has a Significant Role in Mycosis Fungoides (MF) Cell Biology: An RNA Sequencing Study of 40 MF Cases.

The purpose of this RNA sequencing study was to investigate the biological mechanism underlying how the transcription factors (TFs) Twist1 and Zeb1 influence the prognosis of mycosis fungoides (MF). We used laser-captured microdissection to dissect malignant T-cells obtained from 40 skin biopsies from 40 MF patients with stage I-IV disease. Immunohistochemistry (IHC) was used to determinate the protein expression levels of Twist1 and Zeb1. Based on RNA sequencing, principal component analysis (PCA), differential expression (DE) analysis, ingenuity pathway analysis (IPA), and hub gene analysis were performed between the high and low Twist1 IHC expression cases. The DNA from 28 samples was used to analyze the TWIST1 promoter methylation level. In the PCA, Twist1 IHC expression seemed to classify cases into different groups. The DE analysis yielded 321 significant genes. In the IPA, 228 significant upstream regulators and 177 significant master regulators/causal networks were identified. In the hub gene analysis, 28 hub genes were found. The methylation level of TWIST1 promoter regions did not correlate with Twist1 protein expression. Zeb1 protein expression did not show any major correlation with global RNA expression in the PCA. Many of the observed genes and pathways associated with high Twist1 expression are known to be involved in immunoregulation, lymphocyte differentiation, and aggressive tumor biology. In conclusion, Twist1 might be an important regulator in the disease progression of MF.

TWIST1 regulates proliferation, migration, and invasion and is a prognostic marker for oral tongue squamous cell carcinoma.

Epithelial-mesenchymal transition is one of the main mechanisms for tumor progression and metastasis. Transcription factors such as TWIST1 are key regulators of the epithelial-mesenchymal transition and are regarded as potential therapeutic targets for the treatment of cancer. The purpose of this study was to examine TWIST1 as a possible epithelial-mesenchymal transition-related prognostic biomarker in oral epithelial dysplasia and oral tongue squamous cell carcinomas, as well as the biological behavior of TWIST1-silencing in oral tongue squamous cell carcinomas cell lines. Immunohistochemical analysis of TWIST1, E-cadherin, and N-cadherin was carried out in 47 samples representing oral epithelial dysplasia and 41 oral tongue squamous cell carcinomas. The suppression of TWIST1 expression was performed using shRNA-expression vectors in HSC-3 and SCC-9 cells to investigate in vitro the impact of TWIST1 on proliferation, apoptosis, viability, migration, and invasion of SCC-9 and HSC-3 cells. The expression of nuclear TWIST1 was significantly higher in oral tongue squamous cell carcinomas than in oral epithelial dysplasis (p < 0.0001), whereas TWIST1 in the cytoplasm was more expressed in oral epithelial dysplasis (p=0.012). The high cytoplasmic expression of TWIST1 was significantly associated with shortened overall survival (p < 0.05), and increased nuclear TWIST1 expression was significantly related to high risk of recurrence (p=0.03). Knockdown of TWIST1 in oral tongue squamous cell carcinomas cells induced the expression of E-cadherin and inhibited N-cadherin, which were followed by decreased proliferation, migration, and invasion. Our research suggests that TWIST1 is linked to the development of oral tongue carcinogenesis and may be used as a prognostic indicator and therapeutic target for oral tongue squamous cell carcinomas patients.

Expression of Twist 1 in bone marrow and extramedullary lesions of patients with myeloma and its prognostic significance

In patients with multiple myeloma (MM) extramedullary (EM) lesions could be a possible indicator of poor prognosis. Twist 1, an EMT related transcription factor, is known to play an important role in embryonic development and also in the process of tumor cell proliferation, apoptosis, metastasis etc. The Twist 1 protein expression hence has prognostic significance in MM. This study investigates expression of Twist-1 in MM patients with extramedullary lesions and its relationship with clinicopathological data and prognosis, so as to elucidate the clinical significance of Twist-1 expression. Patients with MM complicated with EM lesions were selected retrospectively, including 35 cases in the bone marrow group (MM complicated with extramedullary lesions) and 80 cases in the extramedullary group (MM combined with extramedullary lesions). The expression of Twist 1 protein was detected by immunohistochemistry. The relationship between Twist 1 protein expression and general clinicopathological data was analyzed by statistics. The relationship between Twist 1 protein expression and prognosis was analyzed by COX single factor and multivariate regression analysis. Immunohistochemical staining showed that the expression level of Twist-1 protein in the nucleus of myeloma cells in the extramedullary group was significantly higher than that of the bone marrow group. However, in the cytoplasm of myeloma cells, there was no significant difference in the expression level of Twist-1 protein between the extramedullary group and the bone marrow group. The expression level of Twist-1 protein in extramedullary tissue was not related to sex, age, D-S stage, ISS stage, immunoglobulin type, creatinine and hemoglobin. The expression level of β2-microglobulin in Twist-1 high expression group was significantly higher than that of Twist-1 low expression group. Follow-up findings discovered that overall survival (OS) of Twist 1 high expression group was significantly lower than that of Twist 1 low expression group. COX multivariate analysis showed that Twist 1 protein and International Staging System (ISS) stage were independent risk factors affecting OS for 3 years. The high expression rate of Twist 1 protein in extramedullary lesions of MM patients with extramedullary lesions is significantly higher than that in bone marrow tissues of patients with extramedullary lesions. The results suggest that high expression of Twist 1 protein is an independent risk factor affecting the prognosis of MM patients with extramedullary lesions.

Prognostic and clinicopathological value of Twist expression in esophageal cancer: A meta-analysis.

BACKGROUNDTwist is a repressor of E-cadherin transcription that induces epithelial-mesenchymal transition and cancer metastasis. However, the prognostic value of Twist expression in patients with esophageal cancer remains controversial.AIMTo investigate the prognostic and clinicopathological value of Twist expression in esophageal cancer.METHODSPublished literature in databases such as EMBASE, Web of Science, PubMed, China National Knowledge Infrastructure, Wanfang, and VIP databases was searched for eligible articles. Participants with esophageal cancer whose tumor tissues underwent immunohistochemistry to detect the expression of Twist were considered. Our meta-analysis was conducted using Stata version 12.0. The hazard ratio (HR) and relative ratio (RR) with their 95%CI were pooled. Heterogeneity was estimated by I2 statistics.RESULTSEleven articles published between 2009 and 2021 fulfilled the selection criteria. The pooled HR for overall survival was 1.88 (95%CI: 1.32-2.69, I2 = 68.6%), and the pooled HR for disease-free survival/relapse-free survival/progression-free survival was 1.84 (95%CI: 1.12-3.02, I2 = 67.1%), suggesting that high Twist expression is associated with poor prognosis in esophageal cancer patients. In addition, overexpression of Twist was correlated with T stage (T3 + T4 vs T1 + T2, RR = 1.38, 95%CI: 1.14-1.67), lymph node metastasis (yes vs no, RR = 1.34, 95%CI: 1.11-1.60), distant metastasis (yes vs no, RR = 1.18, 95%CI: 1.02-1.35), tumor, node and metastasis (TNM) stage (III + IV vs I + II, RR = 1.35, 95%CI: 1.14-1.60), and clinical stage (III + IV vs I + II, RR = 1.58, 95%CI: 1.34-1.87). However, no correlation between Twist expression and age, gender, tumor location, differentiation, or venous invasion was observed.CONCLUSIONHigh expression of Twist is associated with poor esophageal cancer prognosis. Moreover, Twist overexpression is correlated with T stage, lymph node metastasis, distant metastasis, TNM stage, and clinical stage, which indicates that Twist might accelerate esophageal cancer progression and metastasis.

High Expression of TWIST-1 in the Chronic Myeloid Leukemia Patients with T315I Mutation

Background: Among the known ABL mutations in chronic myeloid leukemia (CML), T315I is of particular importance. The T315I mutation may develop resistant cells that increase disease progression. TWIST-1 expression is impaired in patients with increased drug resistance. Objectives: The current study aimed to measure the expression of TWIST-1 gene in CML patients to investigate its association with T315I mutation. Methods: Peripheral blood samples were taken from 40 CML patients. The expression of TWIST-1 and BCR-ABL1 genes was quantified by real-time polymerase chain reaction (PCR). The gene expression was evaluated by REST software. cDNA was used for amplification refractory mutation system (ARMS)-PCR reaction. Results: Of the 40 patients (age range: 19 - 72 years) participating in the study, 23 (57.7%) were female, and 17 (42.5%) were male. The expression of TWIST-1 gene was 43 ± 184.09-fold. The T315I mutation was detected in 3 (7.5%) patients. Conclusions: According to our results, the TWIST-1 gene expression in patients with T315I mutation was significantly higher than patients without that mutation.

Normal and cancer fibroblasts differentially regulate TWIST1, TOX and cytokine gene expression in cutaneous T-cell lymphoma

BackgroundMycosis fungoides (MF) is a primary cutaneous T-cell lymphoma (CTCL) that transforms from mature, skin-homing T cells and progresses during the early stages in the skin. The role of the skin microenvironment in MF development is unclear, but recent findings in a variety of cancers have highlighted the role of stromal fibroblasts in promoting or inhibiting tumorigenesis. Stromal fibroblasts are an important part of the cutaneous tumor microenvironment (TME) in MF. Here we describe studies into the interaction of TME-fibroblasts and malignant T cells to gain insight into their role in CTCL.MethodsSkin from normal (n = 3) and MF patients (n = 3) were analyzed for FAPα by immunohistochemistry. MyLa is a CTCL cell line that retains expression of biomarkers TWIST1 and TOX that are frequently detected in CTCL patients. MyLa cells were cultured in the presence or absence of normal or MF skin derived fibroblasts for 5 days, trypsinized to detached MyLa cells, and gene expression analyzed by RT-PCR for MF biomarkers (TWIST1 and TOX), Th1 markers (IFNG, TBX21), Th2 markers (GATA3, IL16), and proliferation marker (MKI67). Purified fibroblasts were assayed for VIM and ACTA2 gene expression. Cellular senescence assay was performed to assess senescence.ResultsMF skin fibroblast showed increased expression of FAP-α with increasing stage compared to normal. Normal fibroblasts co-cultured with MyLa cells suppressed expression of TWIST1 (p < 0.0006), and TOX (p < 0.03), GATA3 (p < 0.02) and IL16 (p < 0.03), and increased expression of IFNG (p < 0.03) and TBX21 (p < 0.03) in MyLa cells. In contrast, MyLa cells cultured with MF fibroblasts retained high expression of TWIST1, TOX and GATA3. MF fibroblasts co-culture with MyLa cells increased expression of IL16 (p < 0.01) and IL4 (p < 0.02), and suppressed IFNG and TBX21 in MyLa cells. Furthermore, expression of MKI67 in MyLa cells was suppressed by normal fibroblasts compared to MF fibroblasts.ConclusionSkin fibroblasts represent important components of the TME in MF. In co-culture model, normal and MF fibroblasts have differential influence on T-cell phenotype in modulating expression of Th1 cytokine and CTCL biomarker genes to reveal distinct roles with implications in MF progression.

Abstract PO-067: The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer

Abstract Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer mortality. Although therapeutic advances have been made, resistance to treatments remain high and the overall survival is still dismal. The high expression of the transcription factor TWIST1 strongly correlates with invasive and metastatic cancers, and is generally attributed to the epithelial-to-mesenchymal transition phenotype. We have demonstrated that TWIST1 can antagonize the induction of fail-safe programs as oncogene (KrasG12D)-induced senescence (OIS) in primary NSCLC tumor. OIS suppression by TWIST1 required increased global O-GlcNAcylation, which perhaps can also impact DNA repair and radiation response. As TWIST1 is essential for development, deciphering the critical domains and downstream transcriptional targets required for pro-tumorigenicity and radioresistance may allow the identification of new therapeutic strategies by targeting TWIST1. We created a transactivation-null TWIST1 mutant, by mutation of phenylalanine 191 to glycine, genetically engineered mouse model (GEMM) utilizing the tetracycline-inducible gene expression system. In these GEMMs, doxycycline treatment allows a concomitant induction of KrasG12D oncogene (R) with TWIST1 (T) or with TWIST1F191G mutant (F) expression, specifically in the lung epithelium directed by CCSP promoter-rtTA (C) transgene. CRT mice presented a more aggressive tumor progression and a shorter survival (median= 15.6 weeks) compared to CR (31 weeks). TWIST1F191G expression in CRF abrogates these effects (26.7 weeks) suggesting that the TWIST1 transactivation domain is required for TWIST1-dependent accelerated tumorigenesis. CRT mice, HBEC, and H460 cells overexpressing TWIST1 showed radiation resistance. A second KrasG12D lung tumor GEMM with induction of TWIST1 prior to 15Gy lung tumor irradiation showed lung tumor stasis compared to regression without TWIST1 expression. Histological analysis showed a strong expression of TWIST1 in CRT lungs while CRF showed a progressive loss over time suggesting that TWIST1F191G was non-functional and conferred no selective advantage. CRT mice also had lung tumors with higher proliferation (by Ki67), reduced apoptosis (by cleaved caspase3) and a decreased cell cycle arrest (by p16) compared to CR lung tumors. In comparison, CRF mice lung tumors did not show any change in cell death but showed increased p16 cell cycle arrest marker suggesting that the transactivation domain of TWIST1 is important for the suppression of OIS. We are exploring similar in vitro phenotypes using a primary immortalized HBEC cell lines co-transfected with HRasG12V oncogene and TWIST1 versus TWIST1 transactivation mutant. In future work, we are investigating the role of the TWIST1 transactivation domain in the induction of O-GlcNAcylation and the stabilization and/or activation of critical targets for OIS suppression and radiation resistance, with the goal of identifying new therapeutic targets and radiosensitizers. Citation Format: Audrey Lafargue, Hailun Wang, Sivarajan T. Chettiar, Rajendra P. Gajula, Caleb Smack, Ismaeel Siddiqui, Kekoa Taparra, Christine Lam, Francesca Carrieri, Katriana Nugent, Natasha Zachara, Phuoc T. Tran. The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-067.

ABCB1 inhibition provides a novel therapeutic target to block TWIST1-induced migration in medulloblastoma.

BackgroundTherapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial–mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis.MethodsA 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA.ResultsMetastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05).ConclusionsIn this study, we identified a TWIST1–ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

MBRS-45. TWIST1 AND ABCB1 ARE FUNCTIONAL DETERMINANTS OF METASTASIS IN MEDULLOBLASTOMA

Paediatric medulloblastomas (MB) are frequently metastatic, resulting in a poor prognosis for the patient. Of the four MB subgroups, group 3 patients present with the highest rates of metastasis and worst outcomes. The mechanisms behind the metastatic process are poorly understood, limiting our ability to develop novel therapeutic treatments. We hypothesised that the epithelial-mesenchymal transition (EMT) transcription factor TWIST1 and the multidrug efflux pump ABCB1 (ATP-binding cassette subfamily B member 1) synergistically drive MB metastasis. TWIST1 protein expression was analysed in patient tissue microarrays by immunohistochemistry. High TWIST1 expression was associated with metastatic patients (p=0.041). Physical and functional interactions between TWIST1 and ABCB1 were investigated using chromatin immunoprecipitation (ChIP) and a 3D migration and invasion model. ChIP analysis confirmed TWIST1 binding to the ABCB1 promoter in SHH (ONS-76) and group 3 (D283MED and HD-MB03) metastatic cell lines. TWIST1 and ABCB1 were inhibited in HDMB03 cells with harmine and vardenafil respectively, resulting in attenuated cell migration in the 3D model. Western blot and qRT-PCR analysis of harmine treated cells confirmed a reduction in ABCB1 protein and gene expression. Overall our data reveals TWIST1 and ABCB1 to be key targets for MB metastatic disease. Using bioinformatics analysis and ChIP sequencing, additional TWIST1 downstream targets are now being identified and compared across the metastatic cell lines (ONS-76, D283MED and HD-MB03). This data will provide a deeper insight into the pathways associated with MB metastases, enabling personalised treatment approaches for patients with metastatic disease.

Magnesium Isoglycyrrhizinate Induces an Inhibitory Effect on Progression and Epithelial-Mesenchymal Transition of Laryngeal Cancer via the NF-κB/Twist Signaling.

BackgroundMagnesium isoglycyrrhizinate (MI) was extracted from roots of the plant Glycyrrhiza glabra, which displays multiple pharmacological activities such as anti-inflammation, anti-apoptosis, and anti-tumor. Here, we aimed to investigate the effect of MI on the progression and epithelial–mesenchymal transition (EMT) of laryngeal cancer.MethodsForty laryngeal cancer clinical samples were used. The role of MI in the proliferation of laryngeal cancer cells was assessed by MTT assay, Edu assay and colony formation assay. The function of MI in the migration and invasion of laryngeal cancer cells was tested by transwell assays. The effect of MI on apoptosis of laryngeal cancer cells was determined by cell apoptosis assay. The impact of MI on tumor growth in vivo was analyzed by tumorigenicity analysis using Balb/c nude mice. qPCR and Western blot analysis were performed to measure the expression levels of gene and protein, respectively.ResultsWe identified that EMT-related transcription factor Twist was significantly elevated in the laryngeal cancer tissues. The expression of Twist was also enhanced in the human laryngeal carcinoma HEP-2 cells compared with that in the primary laryngeal epithelial cells. The high expression of Twist was remarkably correlated with poor overall survival of patients with laryngeal cancer. Meanwhile, our data revealed that MI reduced cell proliferation, migration and invasion and enhanced apoptosis of laryngeal cancer cells in vitro. Moreover, MI decreased transcriptional activation and the expression levels of NF-κB and Twist, and alleviated EMT in vitro and in vivo. MI remarkably inhibited tumor growth and EMT of laryngeal cancer cells in vivo.ConclusionMI restrains the progression of laryngeal cancer and induces an inhibitory effect on EMT in laryngeal cancer by modulating the NF-κB/Twist signaling. Our finding provides new insights into the mechanism by which MI inhibits laryngeal carcinoma development, enriching the understanding of the anti-tumor function of MI.

TWIST1 expression and clinical significance in type I endometrial cancer and premalignant lesions: A retrospective clinical study.

The aim of this study was to assess the correlation of TWIST1 expression with clinical parameters and the prognosis of type I endometrial cancer (EC).This retrospective study enrolled 345 patients. Immunohistochemical staining was performed on 55 normal endometrium (NE) samples, 27 atypical hyperplasia (AH) samples, and 263 type I EC samples. The association between TWIST1 staining and clinical characteristics and survival was evaluated by univariate and multivariate analyses.We found significantly higher TWIST1 expression in patients with AHs and type I ECs than NEs, but there was no significant difference between TWIST1 expression in AHs and type I ECs. Aberrant TWIST1 expression was significantly associated with clinical parameters, indicating poor prognosis and shorter patient survival. Pearsons Chi-Squared test showed that high TWIST1 expression was significantly associated with a shorter disease-free survival and overall survival. More importantly, multivariate analysis showed that high TWIST1 expression, in addition to myometrial invasion, lymph vascular space invasion, and lymph node metastasis, was an independent predictor of worse DFS in patients with type I ECs.Our findings suggest that TWIST1 might be useful in diagnosing ECs and predicting prognosis in patients with AHs and type I ECs.

TWIST1 upregulation affects E-cadherin expression in brain metastases.

E-cadherin is a calcium-dependent glycoprotein whose main role is cell-cell adhesion. Its transcriptional repressor TWIST1 is a basic helix-loop-helix (bHLH) protein that participates in gastrulation and formation of mesodermal tissues during embryogenesis. In adult tissues, the high expression of TWIST1 induces the epithelial-mesenchymal transition (EMT)-a process in which cells become motile and able to metastasize. In this paper, we investigated the involvement of E-cadherin and TWIST1 in the carcinogenesis of brain metastases originating from two different primary sites-breast and lung. The localization and expression of E-cadherin and its transcriptional repressor TWIST1 were investigated using a DAB-labeled streptavidin-horseradish peroxidase immunohistochemical reaction and specific monoclonal antibodies against TWIST1 and E-cadherin. Image J software was used for semi-quantitative analysis while H-score served for statistical evaluations. Immunohistochemistry showed that the expression of E-cadherin was downregulated in 85.7% of brain metastases, while at the same time, 82.2% of them showed upregulated TWIST1. Statistical analysis confirmed a significant negative correlation between expressions of TWIST1 and E-cadherin (p = 0.001). When the brain metastases expression levels were compared to primary breast tumors in corresponding patients, E-cadherin showed higher expression in primary pairs compared to corresponding metastases. Consistent to its role, TWIST1 was downregulated in all primary tumor samples in comparison to corresponding metastases pairs (p = 0.034). This research provides valuable data regarding molecular events involving two EMT key components that could give directions for new possibilities for brain metastases diagnosis and treatment.

Abstract 6060: The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer

Abstract Lung cancer is the most common cause of mortality by cancer. Non-small cell lung carcinoma (NSCLC) accounts for 80% of lung cancers. Although progresses has been realized, resistance to current treatments still remain high and the overall survival is a dismal ~16%. The high expression of TWIST1 in cancers strongly correlates with invasive, resistant, and metastatic cancers, and is attributed to the induction of the epithelial-mesenchymal transition phenotype. Additionally, TWIST1 antagonizes the induction of the oncogene (KrasG12D)-induced senescence in primary NSCLC tumor. As TWIST1 is a pleiotropic transcription factor essential for development, the targeting of the entire TWIST1 protein may cause unintended side effects. Dissecting what are the critical domains of TWIST1 and its crucial downstream transcriptional targets required for TWIST1-dependent radioresistance and pro-tumorigenicity would allow to decipher new therapeutic strategies. In this goal, we generated a transactivation-null TWIST1 mutant by mutation of phenylalanine at position 191 to glycine and aimed to characterize its functions. We generated mouse models utilizing a tetracycline-inducible gene expression system: in this model, treatment with doxycycline allows to control concomitant induction of KrasG12D oncogene expression (R) with TWIST1 (T)/Luciferase expression or with TWIST1F191G mutant (T-F191G)/Luciferase expression, specifically in the lung epithelium using the CCSP promoter-rtTA construct (C). TWIST1 expression in CRT model induced a more aggressive tumor progression and a shorter survival (median= 15,6 weeks) compared to CR model (median= 31 weeks). The expression of TWIST1F191G mutant in CRT-F191G model abrogates these effects (median= 26.7 weeks) showing the importance of the transactivation domain of TWIST1 to fulfill its pro-tumor progression functions. Next, the histological analysis of the lung tumor sections of these models shown that the expression of TWIST1 in CRT mice induced a higher positivity for Ki67 and a reduced positivity for Caspase3. CRT mice also present a decrease for p16 expression compared to CR mice reflecting TWIST1-induced OIS suppression. Additionally, the expression of TWIST1F191G mutant shown a similar profile to CR model potentially indicating a role of TWIST1 transactivation domain in the suppression of OIS. On the other hand, the expression of TWIST1 in NSCLC lines conferred a partial radiation resistance. We also observed that the expression of TWIST1 in AEF line conferred the same resistant profile while the expression of TWIST1F191G increased the sensitivity to radiation. Moreover, the CRT model also presented more resistance to 1 × 15Gy or 10 × 3Gy thoracic irradiation suggesting again that targeting TWIST1 transactivation function would confer a potential new important therapeutic option. Citation Format: Audrey Lafargue, Hailun Wang, Sivarajan T. Chettiar, Rajendra P. Gajula, Kekoa Taparra, Katriana Nugent, Phuoc T. Tran. The transactivation domain of TWIST1 is required for TWIST1-induced aggressiveness in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6060.